All the cells circulating in the blood are descendants of a very small number of pluripotent stem cells. These ancestral cells, which comprise less than 0.01 percent of the nucleated cells in the bone marrow, are capable of restoring normal hematopoiesis in individuals in need of reconstitution of their bone marrow. The pluripotent stem cell has the unique capacity for self-renewal and the potential for growth and differentiation along granulocytic, monocytic, erythroid, megakaryocytic, and lymphoid lineages. Some stem cells divide and give rise to progeny that lose their ability to differentiate along multiple pathways and become committed to a specific hematopoietic lineage. These committed progenitor cells continue to proliferate and differentiate into morphologically identifiable precursor cells which then undergo terminal maturation, thereby developing highly specialized functions and lose their ability to proliferate. Techniques have been developed which support the growth and differentiation of hematopoietic progenitor cells in vitro. Using these techniques, hematopoietic colonies of mixed and single lineages have been identified and characterized with respect to the factors required for their growth.
Mammalian blood cells provide for an extraordinarily diverse range of activities. The blood cells are divided into several lineages based on function. The lymphoid lineage, comprising B cells and T cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. The myeloid lineage, which includes monocytes, granulocytes, megakaryocytes, as well as other cells, monitors for the presence of foreign bodies in the blood stream, provides protection against neoplastic cells, scavenges foreign materials in the blood stream, produces platelets, and the like. The erythroid lineage provides the red blood cells, which act as oxygen carriers.
As noted above, the stem cell population constitutes only a small percentage of the total number of leukocytes in bone marrow. At the present time it is not known how many of the markers associated with differentiated cells are also present on the stem cell. One marker which has been indicated as present on stem cells, CD34, is also found on a significant number of lineage-committed progenitors. Another marker that provides for some enrichment of progenitor activity is Class II HLA (particularly a conserved DR epitope recognized by a monoclonal antibody designated J1-43). However, these markers are found on numerous lineage-committed hematopoietic cells. In particular, B cells (CD19+) and myeloid cells (CD33+) make up 80-90% of the CD34+ population. Moreover, a combination of CD 3, 8, 10, 15, 19, 20 and 33 will mark .ltoreq.90% of all CD34+ cells. Therefore, in view of the small proportion of the total number of cells in the bone marrow which are stem cells, the uncertainty of the markers associated with stem cells, as distinct from more differentiated cells, and the general inability to conduct a definitive biological assay for human stem cells, the identification and purification of stem cells has been elusive.
Aldehyde dehydrogenase (ALDH) is an enzyme responsible for oxidizing intracellular aldehydes and plays an important role in metabolism of ethanol, vitamin A, and cyclophosphamide. Substrates for these enzymes include acetaldehyde and biogenic amines produced during catecholamine catabolism. Further, ALDH appears to be a crucial step in the conversion of vitamin A to its active metabolite, retinoic acid (J. Labrecque, et al, Biochem Cell Biol, 71:85, 1993; A. Yoshida, el al., Enzyme, 46:239, 1992). Of the four ALDH isozymes identified (A. Yoshida, et al., Enzyme, 46:239, 1992), liver cytosolic ALDH appears to be the specific isozyme involved in metabolism of vitamin A (J. Labrecque, et al., Biochem Cell Biol, 71:85, 1993; A. Yoshida, et al., Enzyme, 46:239, 1992) and cyclophosphamide resistance (J. E. Russo et al., Cancer Res 48:2963, 1988). Both hematopoietic progenitors and intestinal crypt stem cells display high levels of cytosolic ALDH, and are accordingly relatively resistant to cyclophosphamide. Although all hematopoietic progenitors are known to express relatively high levels of cytosolic aldehyde dehydrogenase, both mouse and human HSC appear to express significantly higher levels than less primitive hematopoietic progenitors. Primitive hematopoietic progenitors are more resistant to 4HC than are later progenitors, and this appears to result in large part from their high expression of cytosolic ALDH rather than from their non-cycling status. However, there is no difference in the sensitivity to 4HC analogues of primitive hematopoietic progenitors that are not inactivated by aldehyde dehydrogenase (E. A. Sahovic et al., Cancer Res, 48:1223, 1988).
Increased AND-dependent aldehyde dehydrogenase (ALDH, Enzyme Commission 1.2.1.3) activity has been identified as a mechanism of antitumor drug resistance to the alkylating agent cyclosphosphamide (CPA). CPA, a nitrogen mustard derivative incorporating an oxazaphosphorine, is a prodrug, requiring activation by the cytochrome P-450 mixed function oxidase system to produce 4-hydroxycyclophosphamide (4HC). A spontaneous beta elimination from aldophosphamide liberates the active DNA alkylating agent, phosphoramide mustard, plus acrolein.
CPA is currently used clinically in the treatment of a diverse group of solid and hematologic malignancies, and is a key component of the cytoreductive regimens prior to bone marrow transplantation. A major factor in the success of CPA has been the high therapeutic index observed (Mullins and Colvin, Cancer Chemother Repts, 59:411, 1975). This high therapeutic index is due in part to a "sparing" effect that CPA exerts on normal hematopoietic stem cells (Fried, et al., Cancer Research, 37:1205, 1977) and intestinal crypt stem cells. While damage to these rapidly proliferating stem cell renewal systems is the dose-limiting toxicity of other alkylating agents, these tissues are not profoundly effected by CPA. The diminished toxicity is specific for CPA (and its activated congeners) among alkylating agents. Thus, elevated ALDH levels have been postulated as a mechanism for the relative resistance of bone marrow and intestinal stem cells to CPA. In vivo studies in mice have shown that a cytosolic ALDH isozyme found in murine tumor tissue is responsible for conferring cyclophosphamide resistance (J. E. Russo et al., Enzymology and Molecular Biology of Carbonyl Metabolism, 2:65-79, 1989).
Elevated levels of ALDH have been best characterized as a mechanism of cellular resistance to CPA in the L1210 murine lymphocytic leukemia model (DeWys, 1973), in which a 200-fold higher cytosolic ALDH activity was measured in the cell line resistant to CPA (L1210/CPA) compared to the sensitive, wild-type line.
By definition human hematopoietic stem cells (HSC) are progenitor cells and are pluripotent in that they have the ability to repopulate lymphohematopoietic lineages on a long-term basis. The isolation of human HSC remains a goal particularly difficult to achieve, since prior to the present invention no single cell characteristic has been found specific for HSC which could be used to prepare an enriched cellular composition. Therefore, the isolation and/or identification of HSC suitable for re-introduction into a host requires an eventual in vivo approach, yet prior art techniques for HSC detection, namely measurement of substrate oxidation in whole cell lysates (J. E. Russo et al., Cancer Res 48:2963, 1988; J. E. Russo et al., Enzymology and Molecular Biology of Carbonyl Metabolism 2:65-79, 1989) or reaction of fixed cells with antibodies (M. B. Kastan et al., Blood 75:1947, 1990; J. E. Russo et al., Enzymology and Molecular Biology of Carbonyl Metabolism 2:65-79, 1989) are lethal to the cells being studied. By the same token, the applicability of animal models has been limited because analogues of many well-characterized HSC markers have not been identified in other species (G. J. Spangrude et al., Science, 241:58, 1988; C. T. Jordan, et al., Cell, 61:953, 1990). Thus, there is a need for a stem cell enriched composition which is produced using essentially non-toxic methodology thereby allowing safe and effective reconstitution of a host in need of such therapy. The present invention answers this need.